4.4.5.1 Osteoclastogenic pathways in FCD pathogenesis
The increased levels of CTX-I in FCD serum relative to controls is indicative of a net increase of bone resorption activity, however a notable observation was that the RANKL/OPG ratio did not exhibit a relationship with CTX-I levels in synovial fluid or serum. As previously suggested, this is evident of non-canonical osteoclastogenic pathway activity that shares regulation of pathogenic bone remodelling signalling in disease states such as FCD and uOA. Further understanding of the influence of canonical and non-canonical pathways in FCD pathogenesis is critical to developing anti-resorptive therapies such as those used in trials for preserving bone in rheumatoid arthritis (Gamez-Nava et al., 2013).
A key finding within this study was that RANKL expression appears to be predominantly dependent on pro-inflammatory regulators, whereas OPG may be influenced by subchondral bone loading due to its discriminatory ability of FCD and uOA synovial fluid. The canonical bone remodelling pathway is dependent on the activation of receptor activator of NF-κB (RANK) found on the surface of pre and mature osteoclasts, which when activated by RANKL located on activated osteoblasts, osteocytes and some immune cell types, stimulates osteoclast differentiation and bone resorption through intracellular stimulation of the NF-κB pathway (Tat et al., 2008b, Wijenayaka et al., 2011). However, OPG secreted by osteoblasts can inhibit the activity of RANK by binding and deactivating RANKL, thus the relative quantities of RANKL and OPG can provide an index of bone resorption pathway activation (Tat et al., 2009). The dependence of osteoclast activity on the RANK/RANKL system is evident with investigations into RANKL-knockout mice, which suffer from unopposed bone growth due to the lack of osteoclast maturation and activity (Odgren et al., 2003). In normal functioning, the level of RANKL and OPG expression is dependent on a wide variety of signalling promotors and inhibitors, with multiple levels of ligand and receptor activity.
In earlier years it was recognised that key regulators such as parathyroid hormone (Horwood et al., 1998) and pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 locally produced are involved in canonical bone regulation (Hofbauer et al., 1999). Consistent with the positive correlation found of synovial fluid IL-6 and RANKL within this study, it has been established that IL-6 receptor activation on the osteoblast surface stimulates the janus kinase (JAK) and signal transducer and activators of transcription
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(STAT) pathway, leading to an upregulation of RANKL expression (Osta et al., 2014). There has also been previous evidence of negative regulation of the RANKL/OPG system by anti-inflammatory cytokines. One study investigating IL-10-deficient bone-marrow macrophages observed enhanced osteoclastogenesis and bone resorption relative to controls (Liu et al., 2006). In this model, subsequent introduction of IL-10 reversed this effect evident by down-regulated RANKL mRNA expression coupled with enhanced OPG expression. This mechanism may explain the negative correlation between RANKL and IL-10 found within the correlation analysis.
Sclerostin is primarily known for inhibition of osteogenic gene expression through disruption of the Wnt/β-catenin pathway. However, there is evidence to suggest another role in supporting osteocyte-driven osteoclastogenesis (Wijenayaka et al., 2011). Recombinant human sclerostin was found to dose-dependently down-regulate the expression of OPG mRNA and up-regulate RANKL expression in human primary pre- osteocyte culture and in mouse osteocyte-like MLO-Y4 cells. Furthermore, co-culture of MLO-Y4 cells on a ‘bone-like’ substrate with peripheral blood mononuclear cells in the presence of sclerostin resulted in a 7-fold increase in bone resorption activity, which was eliminated by introduction of OPG (Wijenayaka et al., 2011). This is relevant when considering the PCA results within this study that revealed variances representative of sclerostin and RANKL expression, as it further substantiates sclerostin as a key regulator and discriminatory factor of the identified phenotypes. In a review of the RANKL/OPG system, Tat and colleagues have proposed based on previous literature that OA subchondral bone osteoblasts can be divided into two subpopulations defined as ‘low- OA’ or ‘high-OA’ types (Tat et al., 2009). It was suggested that the ‘low-OA’ phenotype presenting high RANKL/OPG ratios and low endogenous PGE2 production promote
subchondral bone loss, whereas the ‘high-OA’ phenotype that present low RANKL/OPG ratios and high PGE2 promote subchondral bone thickening and sclerosis that is often
attributed to later-stage OA (Tat et al., 2009). This theory could accommodate sclerostin as a possible mechanistic ‘switch’ involved in the transition of osteoblast states.
In vitro studies have shown a number of cytokines and growth factors capable of
substituting the effect of RANK to increase osteoclastogenesis of progenitors. These include TNF-α (Mabilleau et al., 2012), IL-6 and IL-8 (Knowles and Athanasou, 2009), which activate NF-κB intracellular signalling through their own surface receptors. This is could explain the relationship between TNF-α and CTX-I levels found within this study, which was not reflected in TNF-α and RANKL associations, inferring that TNF-α activity is independently promoting bone matrix breakdown. The stronger relationship implies
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TNF-α may have a stronger influence on bone resorption in this disease process. This hypothesis is consistent with the correlation between longitudinal changes in TNF-α and CTX-I and implies that the regulation of TNF-α activity may a key mechanism is the switch toward subchondral bone repair.
Glutamatergic signalling may be in part influencing the incongruities between RANKL/OPG ratio values and bone resorption. Reviews of reported transcript and protein expression of bone cell glutamatergic signalling components found that osteoclasts express iGluRs activated by NMDA, AMPA and kainate, mGluRs and EAATs, implying osteoclasts respond to, as well as regulate extracellular glutamate levels (Brakspear and Mason, 2012, Wen et al., 2015). Early in vitro cultures of osteoclasts exposed to the NMDA glutamate receptor antagonist MK-801 revealed downregulation of osteoclast differentiation and resorption activity, evident by the reduced resorption pits in dentine (Peet et al., 1999). A later study substantiated these findings in two individual in vitro models, one using the murine myelomonocytic RAW 264.2 cell line and the other mouse bone marrow cells, by demonstrating that two antagonists of NMDA receptor (NMDAR), MK-801 and AP-5, dose-dependently inhibited osteoclastogenesis, suggesting that osteoclast progenitors require NMDAR activation for differentiation (Merle et al., 2003). These findings are supportive of the correlation between longitudinal changes in serum glutamate and CTX-I levels, equivalent to that found with TNF-α, implying both mechanical and inflammatory components influence of pathological bone resorption.
4.4.5.2 CTX-I as a biomarker of bone resorption
CTX-I is an indicator of the osteoclastic metabolism of collagen type-I, the predominant collagen in bone and meniscal tissues, it is often utilized as a selective marker for bone turnover for the characterisation of human bone pathologies such as osteopenia, osteoporosis and arthritis (Cremers et al., 2008). Consistent with the findings from this study, CTX-I is elevated in animal models of OA development and progression. In a murine post-traumatic OA (PTOA) model simulated by tibial compression overload and ACL rupture, significantly increased serum levels of CTX-I were observed for up to 56 days post-injury relative to unloaded controls (Khorasani et al., 2015). Although correlations were not explored, the same cohort of mice experienced significant trabecular bone loss explanatory of the increased CTX-I levels, as well as subchondral bone thickening, determined by quantitative structural µCT. Another study investigating changes in a canine partial-medial meniscectomy model which developed tibial
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osteochondral lesions similarly showed CTX-I increases which corresponded to worsening histological scoring of subchondral bone and cartilage OA-related changes (Connor et al., 2009). In human OA studies, it has been shown that CTX-I levels appear to be a risk factor for disease progression and increases in serum precede increases biomarkers of cartilage metabolism, reflecting the earlier pathological changes in bone in the disease (Garnero et al., 2005, Attur et al., 2013). However, interestingly the increased bone resorption only appears to be relevant in the presence of progressing OA states, since individuals with ‘non-progressing OA’ characterised by a lack of change in K/L grade do not exhibit abnormal CTX-I levels (Bettica et al., 2002).
Changes in bone have also been recognised in previous studies investigating joint FCDs. One investigation demonstrated that urinary CTX-I levels were in the ‘upper range of normal’ for patients with tibiofemoral FCDs accompanying ACL insufficiency. Furthermore, CTX-I correlated with reduced tibiofemoral subchondral bone area and cartilage volume determined by MRI methods (Streich et al., 2011), reiterating the sensitivity of CTX-I for detecting relevant bone changes. It is thought that loss of bone and dysregulated remodelling often accompanies FCDs in the form of bone marrow lesions (BMLs), focal areas of bone attrition/sclerosis underlying FCDs, which may be a crucial contributor to the aetiology of FCDs due to their association with rapid articular cartilage loss (Xu et al., 2012). BMLs detected and scored by MRI methods have proven to be strong predictors of defect progression, defined by worsening of chondral lesion MRI scores (Dore et al., 2010). Since bone is a highly mechano-responsive tissue, the presence of BMLs in relation to FCD pathogenesis is likely related to abnormal joint loading. In fact, remodelling is influenced by both mechanical and inflammatory pathways in which it is apparent are both involved in FCD pathogenesis (Burr and Gallant, 2012, Cremers et al., 2008).
The findings from this study corroborate with others regarding the utility of CTX-I as a marker of altered bone resorption activity, therefore may be useful in longitudinal studies where FCDs have been diagnosed. However, it is noteworthy that CTX-I as an individual marker lacks selectivity. Due to the substantial number of regulatory and homeostatic processes involved with bone resorption, non-joint specific bone resorption could potentially affect the diagnostic and prognostic utility. Increased acidity of blood due to diet, osteoporosis as a result of menopause as well as levels of physical activity are just some examples of factors that may affect individual differences (Burr and Gallant, 2012, Cremers et al., 2008). The latter example was demonstrated in a study aiming to distinguish between altered CTX-I levels with the effect of increased exercise
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compared to development of OA in horses, who found similar increases in both groups, stressing the importance of considering such factors in interpretations (Frisbie et al., 2008). Notably from this study, synovial fluid CTX-I significantly associated with BMI, suggesting altered body mass alone may reduce the sensitivity of CTX-I in cross-sectional studies. Perhaps CTX-I may not be a reliable individual marker of pathology, but may be better selective in a combinatory panel with other discriminatory markers or in longitudinal studies whereby many mentioned factors remain consistent.
4.4.5.3 COMP as a biomarker of cartilage remodelling
Several independent studies have demonstrated the potential utility of COMP as a diagnostic and prognostic serum biomarker of cartilage structural degradation in knee OA, due to its sensitivity to joint space narrowing and its known abundance in cartilage tissue where its function involves providing stability to the matrix (Attur et al., 2013, Kraus et al., 2017, Sowers et al., 2009, Halasz et al., 2007). In the cross-sectional analysis, it was evident that serum COMP levels did not vary between FCD subjects and controls. Since FCDs are characterised by progressive focal articular cartilage loss, this finding was unexpected and suggests serum COMP may not be selective of cartilage degradation at this stage of pathology. Consistent with these findings, Streich and colleagues (2012) investigating serum COMP concentrations in individuals with ACL-rupture with accompanying FCDs reported levels within the ‘normal’ clinical ranges, with only weak correlations to medial femoral cartilage area detected by MRI (Streich et al., 2011). However, they found a more reliable link between FCD presence and CTX-II levels, as well as a moderate relationship between CTX-II and cartilage volume and subchondral bone area, suggesting CTX-II may be a stronger indicator of cartilage loss in the earlier stages of pathology.
A likely explanation for the lack of differing COMP levels may be related to its sensitivity to joint activity. Andersson and colleagues (2006) showed that serum COMP levels are significantly elevated after 60 minutes of strenuous exercise, which proceed to significantly return to resting levels 60 minutes following rest (Andersson et al., 2006b). Whereas another group independently demonstrated that even a moderate walking activity of 30 minutes could significantly influence serum COMP up to 9.4% above resting levels (Mundermann et al., 2005b). Due to the difficulties in controlling for activity levels within the tested cohort in this study, it is likely that differences in recent activity may have increased individual variances, ultimately obscuring group differences resultant of chondral defect presence. It could be that the elevated COMP levels following activity are
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either related to the anabolic biosynthetic response of chondrocytes following joint activity, or simply the release from cartilage matrix following structural matrix deformation (Attur et al., 2013).
It is noteworthy that COMP levels were significantly increased following microfracture surgery concomitant with decreased sclerostin levels, implying that COMP synthesis may be important to cartilage repair processes. Indeed, previous authors have demonstrated that COMP is involved in fibril formation of collagen type-I and II by catalysing fibrillogenesis with unique organisation, as well as a possible involvement in supporting mediation of aggrecan interaction (Halasz et al., 2007, Chen et al., 2007). Furthermore, it has been recognised that Wnt signalling is actively regulated during repair, whereby it is involved in chondrogenic or osteogenic lineage commitment of mesenchymal stem cells (MSCs), maintenance of a chondrogenic phenotype and matrix synthesis (Yuan et al., 2016). This suggests the upregulation of COMP synthesis by chondrocytes may be regulated by Wnt following the down-regulation of sclerostin. Considering these findings, serum COMP may be a good indicator of cartilage repair activity in the early stages following marrow stimulation techniques.
On the other hand, overstimulation of Wnt signalling in disease states can promote chondrogenic hypertrophic differentiation and apoptosis, leading to accelerated destruction of cartilage (Yuan et al., 2016, Lewiecki, 2014). Furthermore, from a macroscopic perspective, it is well established that bone marrow stimulation techniques generate a mechanically inferior and disorganised ‘fibrocartilage’ in place of the lost hyaline cartilage, therefore increased COMP levels could be representative of the accelerated destruction of neocartilage in response to continued aberrant loading of the joint. Considering these contrasting mechanisms, longer term studies investigating the persistence of serum COMP are required to understand cartilage repair/degenerative mechanisms in the course of recovery to function following surgery.